Assessment of effects of EQ on cancer cells

The effect of EQ on cisplatin’s antitumor activity was evaluated using an ovarian PA-1 cancer cell line (ATCC, CRL-1572) in 96-well plates. 1500 cells were plated in each well in Eagle’s Minimum Essential Medium (ATCC, Catalog No. 30-2003) supplemented with 10%FBS and incubated for 24 hours. First EQ was added followed by cisplatin and cells were cultured for another 24 hours. Cellular ATP levels as a marker of cell numbers were measured using ViaLight Plus kit (Lonza).

To evaluate the impact of ethoxyquin on cisplatin’s ability to reduce tumors in vivo, a nude mouse model was used. Ovarian cancer cell line PA-1 tumor cells suspended in phosphate buffered saline (3 × 10⁶ cells in 150 μL) were injected subcutaneously into adult female nude mice (athymic nude mice, Cat#007850, The Jackson Lab). Once the tumor size reached 5 mm in diameter, animals were randomly assigned to receive cisplatin alone or cisplatin and ethoxyquin. Cisplatin (3.5 mg/kg/day) was given intravenously by tail vein injection once every three days for a total 8 doses and ethoxyquin (750 μg/kg/day) was administrated daily by intraperitoneal injections for 4 weeks. After four weeks, tumors (n = 7 per group) were harvested and weight and size were measured. DRGs and sciatic nerves were collected to examine the levels of DNA-platinum adducts.